Increase in tumour‐infiltrating lymphocytes with regulatory T cell immunophenotypes and reduced ζ‐chain expression in nasopharyngeal carcinoma patients

The pathological significance of the mechanisms of tumour immune-evasion and/or immunosuppression, such as loss of T cell signalling and increase in regulatory T cells (T_regs), has not been well established in the nasopharyngeal carcinoma (NPC) microenvironment. To evaluate the T_reg immunophenotypes in tumour-infiltrating lymphocytes (TILs), we performed a double-enzymatic immunostaining for detection of forkhead box P3 (FoxP3) and other markers including CD4, CD8, and CD25 on 64 NPC and 36 non-malignant nasopharyngeal (NP) paraffin-embedded tissues. Expression of CD3ζ and CD3ε was also determined. The prevalence of CD4⁺FoxP3⁺ cells in CD4⁺ T cells and the ratio of FoxP3⁺/CD8⁺ were increased significantly in NPC compared with those in NP tissues (P < 0·001 and P = 0·025 respectively). Moreover, the ratio of FoxP3⁺/CD25⁺FoxP3⁻ in NPC was significantly lower than that in NP tissues (P = 0·005), suggesting an imbalance favouring activated phenotype of T cells in NPC. A significant negative correlation between the abundance of FoxP3⁺ and CD25⁺FoxP3⁻ cells (P < 0·001) was also identified. When histological types of NPC were considered, a lower ratio of FoxP3⁺/CD25⁺FoxP3⁻ was found in non-keratinizing and undifferentiated carcinomas. Increased CD4⁺FoxP3⁺/CD4⁺ proportion and FoxP3⁺/CD8⁺ ratio were associated with keratinizing squamous cell carcinoma. A reduced expression of CD3ζ in TILs was found in 20·6% of the NPC tissues but none of the NP tissues. These data provide evidence for the imbalances of T_reg and effector T cell phenotypes and down-regulation of signal-transducing molecules in TILs, supporting their role in suppression of immune response and immune evasion of NPC.     

Low expression of CD39+/CD45RA+ on regulatory T cells (Treg) cells in type 1 diabetic children in contrast to high expression of CD101+/CD129+ on Treg cells in children with coeliac disease

Type 1 diabetes (T1D) and coeliac disease are both characterized by an autoimmune feature. As T1D and coeliac disease share the same risk genes, patients risk subsequently developing the other disease. This study aimed to investigate the expression of T helper (Th), T cytotoxic (Tc) and regulatory T cells (T_reg) in T1D and/or coeliac disease children in comparison to healthy children. Subgroups of T cells (Th : CD4⁺ or Tc : CD8⁺); naive (CD27⁺CD28⁺CD45RA⁺CCR7⁺), central memory (CD27⁺CD28⁺CD45RA⁻CCR7⁺), effector memory (early differentiated; CD27⁺CD28⁺CD45RA⁻CCR7⁻ and late differentiated; CD27⁻CD28⁻CD45RA⁻CCR7⁻), terminally differentiated effector cells (TEMRA; CD27⁻CD28⁻CD45RA⁺CCR7⁻) and T_reg (CD4⁺CD25⁺FOXP3⁺CD127⁻) cells, and their expression of CD39, CD45RA, CD101 and CD129, were studied by flow cytometry in T1D and/or coeliac disease children or without any of these diseases (reference group). Children diagnosed with both T1D and coeliac disease showed a higher percentage of TEMRA CD4⁺ cells (P < 0·05), but lower percentages of both early and late effector memory CD8⁺ cells (P < 0·05) compared to references. Children with exclusively T1D had lower median fluorescence intensity (MFI) of forkhead box protein 3 (FoxP3) (P < 0·05) and also a lower percentage of CD39⁺ and CD45RA⁺ within the T_reg population (CD4⁺CD25⁺FOXP3⁺CD127⁻) (P < 0·05). Children with exclusively coeliac disease had a higher MFI of CD101 (P < 0·01), as well as a higher percentage of CD129⁺ (P < 0·05), in the CD4⁺CD25^hi lymphocyte population, compared to references. In conclusion, children with combined T1D and coeliac disease have a higher percentage of differentiated CD4⁺ cells compared to CD8⁺ cells. T1D children show signs of low CD39⁺/CD45RA⁺ T_reg cells that may indicate loss of suppressive function. Conversely, children with coeliac disease show signs of CD101⁺/CD129⁺ T_reg cells that may indicate suppressor activity.     

Regulatory T cells and chronic immune activation in human immunodeficiency virus 1 (HIV-1)-infected children

The function of CD4⁺ T cells with regulatory activity (T_regs) is the down-regulation of immune responses. This suppressive activity may limit the magnitude of effector responses, resulting in failure to control human immunodeficiency virus 1 (HIV-1) infection, but may also suppress chronic immune activation, a characteristic feature of HIV-1 disease. We evaluated the correlation between viral load, immune activation and T_regs in HIV-1-infected children. Eighty-nine HIV-1-infected children (aged 6–14 years) were included in the study and analysed for HIV-1 plasmaviraemia, HIV-1 DNA load, CD4 and CD8 cell subsets. T_reg cells [CD4⁺ CD25^highCD127^lowforkhead box P3 (FoxP3^high)] and CD8-activated T cells (CD8⁺CD38⁺) were determined by flow cytometry. Results showed that the number of activated CD8⁺CD38⁺ T cells increased in relation to HIV-1 RNA plasmaviraemia (r = 0·403, P < 0·0001). The proportion of T_regs also correlated positively with HIV-1 plasmaviraemia (r = 0·323, P = 0·002), but correlated inversely with CD4⁺ cells (r = −0·312, P = 0·004), thus suggesting a selective expansion along with increased viraemia and CD4⁺ depletion. Interestingly, a positive correlation was found between the levels of T_regs and CD8⁺CD38⁺ T cells (r = 0·305, P = 0·005), and the percentage of T_regs tended to correlate with HIV-1 DNA load (r = 0·224, P = 0·062). Overall, these findings suggest that immune activation contributes to the expansion of T_reg cells. In turn, the suppressive activity of T_regs may impair effector responses against HIV-1, but appears to be ineffective in limiting immune activation.     

Different immunosuppressive mechanisms in multi‐drug‐resistant tuberculosis and non‐tuberculous mycobacteria patients

Previous studies have demonstrated that cells from both multi-drug-resistant tuberculosis (MDR-TB) and non-tuberculous mycobacteria (NTM) patients respond poorly to mycobacterial antigens in vitro. In the present study, we compared the in vitro response of cells isolated from sensitive TB (NR-TB)-, MDR-TB- and NTM-infected patients. Analysis of T cell phenotype ex vivo revealed that both MDR-TB and NTM patients present an increased percentage of CD4⁺CD25^+- forkhead box protein 3 (FoxP3)⁺ and CD4⁺CD25⁺CD127⁻ regulatory T (T_reg) cells when compared to NR-TB. Increased numbers of T_reg cells and interleukin (IL)-10 serum levels were detected in MDR-TB, whereas elevated serum transforming growth factor (TGF)-β was found in the NTM group. Cells of MDR-TB patients stimulated with early secretory antigenic target (ESAT)-6, but not purified protein derivative (PPD), showed a lower frequency of CD4⁺/interferon (IFN)-γ⁺ T cells and enhanced CD4⁺CD25⁺FoxP3⁺, CD4⁺CD25⁺CD127⁻ and CD4⁺CD25⁺IL-10⁺ T cell population. In addition, increased IL-10 secretion was observed in cultured MDR-TB cells following ESAT-6 stimulation, but not in NR-TB or NTM patients. In vitro blockade of IL-10 or IL-10Rα decreased the CD4⁺CD25⁺FoxP3⁺ frequencies induced by ESAT-6 in MDR-TB, suggesting a role of IL-10 on impaired IFN-γ responses seen in MDR-TB. Depletion of CD4⁺CD25⁺ T lymphocytes restored the capacity of MDR-TB T cells to respond to ESAT-6 in vitro, which suggests a potential role for T_reg/T regulatory 1 cells in the pathogenesis of MDR-TB. Together, our results indicate that although the similarities in chronicity, NTM- and MDR-TB-impaired antigenic responses involve different mechanisms.     

Short‐term anti‐CD25 monoclonal antibody administration down‐regulated CD25 expression without eliminating the neogenetic functional regulatory T cells in kidney transplantation

CD4⁺CD25⁺ forkhead box P3 (FoxP3)⁺regulatory T (T_reg) cells are generated and play a key role in the induction and maintenance of transplant tolerance in organ recipients. It has been proposed that interleukin (IL)-2/IL-2 receptor (IL-2R) signalling was essential for the development and proliferation of antigen-activated T cells that included both effector T cells and T_reg cells. Basiliximab (Simulect™), a chimeric monoclonal antibody directed against the α-chain of the IL-2R (CD25), can be expected to not only affect alloreactive effector T cells, but also reduce the number and function of T_reg cells. We therefore examined the effect of basiliximab induction therapy on the number and function of the T_reg cells in renal recipients. Basiliximab decreased the percentage of CD4⁺CD25⁺T cells, but failed to influence the percentage of CD4⁺FoxP3⁺ T_reg cells. The cellular CD25 expression was decreased significantly by basiliximab injection, but CD4⁺CD25⁺ T cells was not depleted from the circulating pool through monoclonal antibody activation-associated apoptosis. Functional analysis revealed that inhibitory function of T_reg cells from recipients with basiliximab injection was not significantly different from recipients without injection. These data indicate that the functional T_reg population may not be influenced by short-term basiliximab treatment.     

Alterations in immune cell subsets and their cytokine secretion profile in childhood idiopathic thrombocytopenic purpura (ITP)

Immune thrombocytopenic purpura (ITP) is acquired autoimmune disease in children characterized by the breakdown of immune tolerance. This work is designed to explore the contribution of different lymphocyte subsets in acute and chronic ITP children. Imbalance in the T helper type 1 (Th1)/Th2 cytokine secretion profile was investigated. The frequency of T (CD3⁺, CD4⁺, CD8⁺) and B (CD19⁺) lymphocytes, natural killer (NK) (CD16⁺56⁺) and regulatory T (T_reg) [CD4⁺CD25^+highforkhead box protein 3 (FoxP3)⁺] cells was investigated by flow cytometry in 35 ITP children (15 acute and 20 chronic) and 10 healthy controls. Plasma levels of Th1 cytokines [interferon (IFN-γ) and tumour necrosis factor (TNF-α)] and Th2 [interleukin (IL)-4, IL-6 and IL-10)] cytokines were measured using enzyme-linked immunosorbent assay (ELISA). The percentage of T_reg (P < 0·001) and natural killer (NK) (P < 0·001) cells were significantly decreased in ITP patients compared to healthy controls. A negative correlation was reported between the percentage of T_reg cells and development of acute (r = −0·737; P < 0·01) and chronic (r = −0·515; P < 0·01) disease. All evaluated cytokines (IFN-γ, TNF-α, IL-4, IL-6 and IL-10) were elevated significantly in ITP patients (P < 0·001, P < 0·05, P < 0·05, P < 0·05 and P < 0·001, respectively) compared to controls. In conclusion, our data shed some light on the fundamental role of immune cells and their related cytokines in ITP patients. The loss of tolerance in ITP may contribute to the dysfunction of T_regs. Understanding the role of T cell subsets will permit a better control of autoimmunity through manipulation of their cytokine network.     

n‐Butyrate Anergized Effector CD4+ T Cells Independent of Regulatory T cell Generation or Activity

CD4⁺ T cell anergy reflects the inability of CD4⁺ T cells to respond functionally to antigenic stimulation through proliferation or IL‐2 secretion. Histone deacetylase (HDAC) inhibitors have been shown to induce anergy in antigen‐activated CD4⁺ T cells. However, questions remain if HDAC inhibitors mediate anergy through direct action upon activated CD4⁺ T cells or through the generation and/or enhancement of regulatory T (T_reg) cells. To assess if HDAC inhibitor n‐butyrate induces anergy independent of the generation or expansion of FoxP3⁺ T_reg cells in vitro, we examine n‐butyrate‐treated murine CD4⁺ T cells for anergy induction and FoxP3⁺ T_reg activity. Whereas n‐butyrate decreases CD4⁺ T cell proliferation and IL‐2 secretion, n‐butyrate did not augment FoxP3 protein production or confer a suppressive phenotype upon CD4⁺ T cells. Collectively, these data suggest that HDAC inhibitors can facilitate CD4⁺ T cell functional unresponsiveness directly and independently of T_reg cell involvement.     

In‐vitro effect of pembrolizumab on different T regulatory cell subsets

Programmed death‐1 (PD‐1) and interactions with PD‐ligand 1 (PD‐L1) play critical roles in the tumour evasion of immune responses through different mechanisms, including inhibition of effector T cell proliferation, reducing cytotoxic activity, induction of apoptosis in tumour‐infiltrating T cells and regulatory T cell (T_reg) expansion. Effective blockade of immune checkpoints can therefore potentially eliminate these detrimental effects. The aim of this study was to investigate the effect of anti‐PD‐1 antibody, pembrolizumab, on various T_reg subpopulations. Peripheral blood mononuclear cells (PBMC) from healthy donors (HD) and primary breast cancer patients (PBC) were treated in vitro with pembrolizumab, which effectively reduced PD‐1 expression in both cohorts. We found that PD‐1 was expressed mainly on CD4⁺CD25⁺ T cells and pembrolizumab had a greater effect on PD‐1 expression in CD4⁺CD25^? T cells, compared to CD4⁺CD25⁺ cells. In addition, pembrolizumab did not affect the expression levels of T_reg‐related markers, including cytotoxic T lymphocyte antigen‐4 (CTLA‐4), CD15s, latency‐associated peptide (LAP) and Ki‐67. Moreover, we report that CD15s is expressed mainly on forkhead box P3 (FoxP3)^?Helios⁺ T_reg in HD, but it is expressed on FoxP3⁺Helios^? T_reg subset in addition to FoxP3^?Helios⁺ T_reg in PBC. Pembrolizumab did not affect the levels of FoxP3^+/?Helios^+/? T_reg subsets in both cohorts. Taken together, our study suggests that pembrolizumab does not affect T_reg or change their phenotype or function but rather blocks signalling via the PD‐1/PD‐L1 axis in activated T cells.     

Imbalance of different types of CD4+forkhead box protein 3 (FoxP3)+ T cells in patients with new‐onset systemic lupus erythematosus

The aim of this study was to examine the numbers of CD4⁺CD25⁻forkhead box protein 3 (FoxP3)⁺, CD4⁺CD25⁺FoxP3⁺ and CD4⁺CXCR5⁺FoxP3⁺ T cells in patients with new-onset systemic lupus erythematosus (SLE). The numbers of CD4⁺CD25⁻FoxP3⁺, CD4⁺CD25⁺FoxP3⁺ and CD4⁺CXCR5⁺FoxP3⁺ T cells and the concentrations of serum interleukin (IL)-10 in 23 patients and 20 healthy controls (HC) were measured. The potential correlations between CD4⁺FoxP3⁺ T cells, serum IL-10 and clinical measures in SLE patients were analysed. In comparison with that in the HC, significantly reduced numbers of CD4⁺CD25⁺FoxP3⁺ and CD4⁺CXCR5⁺FoxP3⁺ T cells, but increased numbers of CD4⁺CD25⁻FoxP3⁺ T cells, were detected, accompanied by significantly lower levels of serum IL-10 in the patients. Stratification analysis indicated the numbers of CD4⁺CD25⁺FoxP3⁺ and CD4⁺CXCR5⁺FoxP3⁺ T cells and serum IL-10 levels in the patients with seropositive anti-dsDNA were significantly less than that in those with seronegative anti-dsDNA. Treatment with the anti-SLE therapy, particularly with prednisone, leflunomide and methotrexate, significantly improved the imbalance of these types of FoxP3⁺ T cells and increased the concentrations of serum IL-10 in the drug-responding patients. The numbers of CD4⁺CD25⁺FoxP3⁺ T cells were correlated negatively with the values of SLE disease activity index (SLEDAI), whereas the numbers of CD4⁺CD25⁻FoxP3⁺ T cells were correlated positively with the values of SLEDAI, erythrocyte sedimentation rate (ESR) and serum C3. In addition, the concentrations of serum IL-10 were correlated positively with the numbers of CD4⁺CD25⁺FoxP3⁺ T cells, but negatively with the values of SLEDAI, serum C3, CRP and ESR in these patients. Our data indicate that the imbalance of different types of FoxP3⁺CD4⁺ T cells may contribute to the development of SLE in Chinese patients.     

The calcineurin inhibitor tacrolimus allows the induction of functional CD4+CD25+ regulatory T cells by rabbit anti-thymocyte globulins

Rabbit anti-thymocyte globulins (rATG) induce CD4⁺CD25⁺forkhead box P3 (FoxP3⁺) regulatory T cells that control alloreactivity. In the present study, we investigated whether rATG convert T cells into functional CD4⁺CD25⁺FoxP3⁺CD127^−/low regulatory T cells in the presence of drugs that may hamper their induction and function, i.e. calcineurin inhibitors. CD25^neg T cells were stimulated with rATG or control rabbit immunoglobulin G (rIgG) in the absence and presence of tacrolimus for 24 h. Flow cytometry was performed for CD4, CD25, FoxP3 and CD127 and the function of CD25⁺ T cells was examined in suppression assays. MRNA expression profiles were composed to study the underlying mechanisms. After stimulation, the percentage CD4⁺CD25⁺FoxP3⁺CD127^−/low increased (from 2% to 30%, mean, P < 0·01) and was higher in the rATG samples than in control rIgG samples (2%, P < 0·01). Interestingly, FoxP3⁺T cells were also induced when tacrolimus was present in the rATG cultures. Blockade of the interleukin (IL)-2 pathway did not affect the frequency of rATG-induced FoxP3⁺ T cells. The rATG tacrolimus-induced CD25⁺ T cells inhibited proliferative responses of alloantigen-stimulated effector T cells as vigorously as rATG-induced and natural CD4⁺CD25⁺FoxP3⁺CD127^−/low T cells (67% ± 18% versus 69% ± 16% versus 45% ± 20%, mean ± standard error of the mean, respectively). At the mRNA-expression level, rATG-induced CD25⁺ T cells abundantly expressed IL-10, IL-27, interferon (IFN)-γ, perforin and granzyme B in contrast to natural CD25⁺ T cells (all P = 0·03), while FoxP3 was expressed at a lower level (P = 0·03). These mRNA data were confirmed in regulatory T cells from kidney transplant patients. Our findings demonstrate that tacrolimus does not negatively affect the induction, phenotype and function of CD4⁺CD25⁺ T cells, suggesting that rATG may induce regulatory T cells in patients who receive tacrolimus maintenance therapy.     

Rapamycin has suppressive and stimulatory effects on human plasmacytoid dendritic cell functions

Plasmacytoid dendritic cells (PDC) are involved in innate immunity by interferon (IFN)-α production, and in adaptive immunity by stimulating T cells and inducing generation of regulatory T cells (T_reg). In this study we studied the effects of mammalian target of rapamycin (mTOR) inhibition by rapamycin, a commonly used immunosuppressive and anti-cancer drug, on innate and adaptive immune functions of human PDC. A clinically relevant concentration of rapamycin inhibited Toll-like receptor (TLR)-7-induced IFN-α secretion potently (−64%) but TLR-9-induced IFN-α secretion only slightly (−20%), while the same concentration suppressed proinflammatory cytokine production by TLR-7-activated and TLR-9-activated PDC with similar efficacy. Rapamycin inhibited the ability of both TLR-7-activated and TLR-9-activated PDC to stimulate production of IFN-γ and interleukin (IL)-10 by allogeneic T cells. Surprisingly, mTOR-inhibition enhanced the capacity of TLR-7-activated PDC to stimulate naive and memory T helper cell proliferation, which was caused by rapamycin-induced up-regulation of CD80 expression on PDC. Finally, rapamycin treatment of TLR-7-activated PDC enhanced their capacity to induce CD4⁺forkhead box protein 3 (FoxP3)⁺ regulatory T cells, but did not affect the generation of suppressive CD8⁺CD38⁺lymphocyte activation gene (LAG)-3⁺ T_reg. In general, rapamycin inhibits innate and adaptive immune functions of TLR-stimulated human PDC, but enhances the ability of TLR-7-stimulated PDC to stimulate CD4⁺ T cell proliferation and induce CD4⁺FoxP3⁺ regulatory T cell generation.     

Significant augmentation of regulatory T cell numbers occurs during the early neonatal period

Regulatory T cells (T_regs) control immune responses by suppressing various inflammatory cells. T_regs in newborn babies may play an important role in preventing excessive immune responses during their environmental change. We examined the number and phenotype of T_regs during the neonatal period in 49 newborn babies. T_regs were characterized by flow cytometry using cord blood (CB) and peripheral blood (PB) from the early (7–8 days after birth) and late (2–4 weeks after birth) neonatal periods. CD4⁺forkhead box protein 3 (FoxP3⁺) T cells were classified into resting T_regs (CD45RA⁺FoxP3^low), activated T_regs (CD45RA^– FoxP3^high) and newly activated T cells (CD45RA^– FoxP3^low). Compared with CB and PB during the late neonatal period, the percentage of T_regs and all T_reg subpopulations in the CD4⁺ lymphocyte population were increased significantly during the early neonatal period. Furthermore, the proportion and absolute number of activated T_regs were increased markedly compared with other T_reg subpopulations, such as resting T_regs and newly activated T cells (non‐T_regs), in the early neonatal period. Increased T_regs concomitantly expressed the suppressive molecule cytotoxic T lymphocyte antigen‐4 (CTLA‐4). The up‐regulated expression of chemokine receptor 4 (CCR4) and down‐regulated expression of CCR7 were also observed in expanded T_regs. When cord blood cells were cultured in vitro with CD3 monoclonal antibodies (mAb) for 5 days, CD4⁺CD45RA^–FoxP3^high cells were increased significantly during the culture. Thus, the presence of increased activated T_regs in early neonates may play an important role in immunological regulation by suppressing excessive T cell activation caused by the immediate exposure to ubiquitous antigens after birth.     

Peripherally Circulating CD4(+) FOXP3(+) CXCR3(+) T Regulatory Cells Correlate with Renal Allograft Function

Peripheral immunoregulation depends on T regulatory cell trafficking into the allograft to modulate the local alloresponse. Little is known about the relevance of trafficking receptors for Tregs after solid organ transplantation in humans. In this study, expression of the peripheral chemokine receptors CXCR3 and CCR5 on CD4⁺ FOXP3⁺ Treg cells was analysed and correlated with allograft function in renal transplant recipients. Flow cytometry analysis of peripheral blood mononuclear cells of 54 renal transplant recipients receiving a calcineurin inhibitor‐based immunosuppression was performed for CD4, CD25, FOXP3, CXCR3 and CCR5 within the first 18 months post‐transplantation. Correlation analysis of chemokine receptor expression and glomerular filtration rate as calculated by MDRD (eGFR) was performed. Expression of the peripheral homing receptors CXCR3 (r = 0.44, P < 0.05) and CCR5 (r = 0.45, P < 0.05) on FOXP3⁺ Tregs correlated with renal allograft function (eGFR) in patients receiving tacrolimus (n = 28), but not cyclosporine A (CsA) (n = 26). CsA but not tacrolimus reduced surface expression of CXCR3 on FOXP3⁺ Tregs in renal transplant recipients as correlated to trough levels (r = ?0.42, P < 0.05). In contrast to CD4⁺ CXCR3⁺ CD25^lo T cells, flow‐sorted CD4⁺ CXCR3⁺ CD25^hi Tregs isolated from healthy individuals did not produce IFNγ or IL‐17 ex vivo and expressed high levels of GARP mRNA both at baseline as well as after TCR activation indicating functional regulatory activity. Expression of the peripheral trafficking receptors CXCR3 and CCR5 on FOXP3⁺ Tregs is associated with renal allograft function. These results suggest that Treg trafficking may also depend on the interaction of CXCR3 or CCR5 and their respective ligands.     

FOXP3 expression and frequency of regulatory T cells in healed individuals from Leishmania major infection and the asymptomatic cases

Two groups of residents in an endemic area of Leishmania major infection in Iran with positive leishmanin skin tests who were either asymptomatic or had healed cutaneous leishmaniasis lesions were compared with respect to their T helper responses. The percentages of regulatory T cells (T_reg; CD4⁺CD25^high FoxP3⁺) from the peripheral blood and CD4⁺ T cells producing intracellular cytokines (IL-4, IL-10, IL-17 and IFN-γ) from the stimulated PBMCs were evaluated by flow cytometry and the expressions of RORC and FOXP3 genes were quantified by real-time RT-PCR. T responder (CD4⁺CD25⁻) and T_reg-enriched (CD4⁺CD25⁺) cells were isolated magnetically and the suppressive capacity of the latter and the cytokines (IFN-γ, TGF-β and IL-10) secreted from them were evaluated by in vitro assays. The results showed that the frequency of T_reg in the studied groups were similar and T_reg from both groups exhibited high yet similar suppressive capacities while significantly higher levels of FOXP3 expression was observed in the asymptomatic group. Taken together, similar frequency and suppressiveness of T_reg combined with high ratios of IFN-γ/IL-10 producing CD4⁺ T cells were common in both groups; however the members of the asymptomatic group appeared to require higher expression of FOXP3 to maintain their immunity to re-infection.     

Regulatory T‐cells in acute dengue viral infection

Although regulatory T‐cells (T_regs) have been shown to be expanded in acute dengue, their role in pathogenesis and their relationship to clinical disease severity and extent of viraemia have not been fully evaluated. The frequency of T_regs was assessed in 56 adult patients with acute dengue by determining the proportion of forkhead box protein 3 (FoxP3) expressing CD4⁺ CD25⁺T‐cells (FoxP3⁺ cells). Dengue virus (DENV) viral loads were measured by quantitative real‐time polymerase chain reaction (PCR) and DENV‐specific T‐cell responses were measured by ex‐vivo interferon (IFN)‐γ enzyme‐linked immunospot (ELISPOT) assays to overlapping peptide pools of DENV‐NS3, NS1 and NS5. CD45RA and CCR4 were used to phenotype different subsets of T‐cells and their suppressive potential was assessed by their expression of cytotoxic T lymphocyte‐antigen 4 (CTLA‐4) and Fas. While the frequency of FoxP3⁺ cells in patients was significantly higher (P < 0·0001) when compared to healthy individuals, they did not show any relationship with clinical disease severity or the degree of viraemia. The frequency of FoxP3⁺ cells did not correlate with either ex‐vivo IFN‐γ DENV‐NS3‐, NS5‐ or NS1‐specific T‐cell responses. FoxP3⁺ cells of patients with acute dengue were predominantly CD45RA⁺ FoxP3^low, followed by CD45RA‐FoxP3^low, with only a small proportion of FoxP3⁺ cells being of the highly suppressive effector Treg subtype. Expression of CCR4 was also low in the majority of T‐cells, with only CCR4 only being expressed at high levels in the effector T_reg population. Therefore, although FoxP3⁺ cells are expanded in acute dengue, they predominantly consist of naive T_regs, with poor suppressive capacity.     

Down syndrome, autoimmunity and T regulatory cells

Autoimmune diseases are more represented in Down syndrome (DS) individuals compared to chromosomally normal people. Natural T regulatory cells (nT_reg) have been considered to be primary in the role of controlling the intensity and targets of the immune response. We have investigated the phenotypical and functional alteration of nT_reg in a group of DS people. The phenotypical characteristic of T_reg cells of 29 DS was analysed and compared with an age‐matched healthy control group. The inhibitory potential of CD4⁺CD25^highCD127^low T regulatory cells was evaluated on autologous CD4⁺CD25^‐ T cell proliferation in response to activation with a mytogenic pan‐stimulus (anti‐CD2, anti‐CD3 and anti‐CD28 antibodies). The CD4⁺CD25^high cells in the DS and control groups were 2·692 ± 0·3808%, n = 29 and 1·246 ± 0·119, n = 29%, respectively (P = 0.0007), with a percentage of forkhead box protein 3 (FoxP3)‐expressing cells of 79·21 ± 3·376%, n = 29 and 59·75 ± 4·496%, respectively (P = 0.0015). CD4⁺CD25⁺FoxP3⁺ cells were increased in peripheral blood from DS subjects (DS mean 5·231 ± 0·6065% n = 29, control mean 3·076 ± 0·3140% n = 29). The majority of CD4⁺CD25^high were CD127^low and expressed a high percentage of FoxP3 (natural T_reg phenotype). While the proliferative capacity of DS T cells was not altered significantly compared to normal individuals, a reduced inhibitory potential of T_reg compared to healthy controls was clearly observed (mean healthy control inhibition in T_eff : T_reg 1:1 co‐culture: 58·9% ± 4·157%, n = 10 versus mean DS inhibition in T_eff : T_reg 1:1 co‐culture: 39·8 ± 4·788%, n = 10, P = 0.0075; mean healthy control inhibition in T_eff : T_reg 1:0·5 co‐culture: 45·10 ± 5·858%, n = 10 versus DS inhibition in T_eff : T_reg 1:0·5 co‐culture: 24·10 ± 5·517%, n = 10, P = 0.0177). DS people present an over‐expressed peripheral nT_reg population with a defective inhibitory activity that may partially explain the increased frequency of autoimmune disease.     

Critical roles of regulatory B and T cells in helminth parasite-induced protection against allergic airway inflammation

The prevalence of allergic asthma and incidences of helminth infections in humans are inversely correlated. Although experimental studies have established the causal relation between parasite infection and allergic asthma, the mechanism of the parasite-associated immunomodulation is not fully elucidated. Using a murine model of asthma and nematode parasite Heligmosomoides polygyrus, we investigated the roles of regulatory B cells (B_reg) and T cells (T_reg) in mediation of the protection against allergic asthma by parasite. H. polygyrus infection significantly suppressed ovalbumin (OVA)-induced allergic airway inflammation (AAI) evidenced by alleviated lung histopathology and reduced numbers of bronchoalveolar inflammatory cell infiltration, and induced significant responses of interleukin (IL)-10⁺ B_reg, IL-10⁺ T_reg and forkhead box protein 3 (FoxP3)⁺ T_reg in mesenteric lymph node and spleen of the mice. Adoptive transfer of IL-10⁺ B_reg and IL-10⁺ T_reg cell prevented the lung immunopathology in AAI mice. Depletion of FoxP3⁺ T_reg cells in FoxP3-diphtheria toxin (DT) receptor transgenic mice by diphtheria toxin (DT) treatment exacerbated airway inflammation in parasite-free AAI mice and partially abrogated the parasite-induced protection against AAI. IL-10⁺ B_reg cells were able to promote IL-10⁺ T_reg expansion and maintain FoxP3⁺ T_reg cell population. These two types of T_regs failed to induce CD19⁺ B cells to transform into IL-10⁺ B_reg cells. These results demonstrate that B_reg, IL-10⁺ T_reg and FoxP3⁺ T_reg cells contribute in A discrepant manner to the protection against allergic airway immunopathology by parasiteS. B_reg cell might be a key upstream regulatory cell that induces IL-10⁺ T_reg response and supports FoxP3⁺ T_reg cell population which, in turn, mediate the parasite-imposed immunosuppression of allergic airway inflammation. These results provide insight into the immunological relationship between parasite infection and allergic asthma.